The isolation and characterization of DNA-binding proteins which function as regulators of genetic processes is a central theme in molecular biological research. Since the vast majority of these protein factors recognize and bind to highly specific DNA sequences, this property has frequently been exploited as a means for identification of the target protein in exploratory assays. Disclosed herein is the development of a class of reagents (see FIG. 1) which are expected to enhance the utility of this approach, by acting as efficient and specific reversible DNA-protein cross-linkers. The reagents exhibit utility in the isolation of a wide variety of DNA-binding proteins. Furthermore, a major application of the cross-linking reagents of the present application will be in the study of proteins which naturally bind metal ions as an integral part of their function. Examples of these include the metalloregulatory proteins (O'Halloran, 1989), and proteins which bind to DNA via the zinc finger (Berg, 1989; Klug, 1989) and related motifs.
Reversible crosslinking of transcription factors to their DNA recognition sequences is expected to greatly facilitate the identification of this important class of proteins (Chu and Orgel, 1992). Incorporation of two discrete metal centers into a bifunctional reagent is intended to enhance the specificity of the reagent for mediation of protein-DNA crosslinks relative to non-productive side-reactions such as DNA-DNA crosslinking, which occurs extensively with simple complexes such as trans-DDP (Ciccarelli et al, 1985; Sherman & Lippard, 1987). The reagent is expected to be especially effective in the crosslinking of transcription factors that bind to DNA via metallopeptide structures such as the `copper fist` of the ACE 1 (CUP1) metalloregulatory protein, (refs **Hamer & Karin) and the zinc-finger motifs found extensively in eukaryotic transcription factors. (Rebek & Nemeth, 1985;, Waters et al., 1978).
Crosslinking strategies have found wide application in studies of the interactions between biological molecules (Angelov, 1988; Czichos et al, 1989; Meffert & Dose, 1988; Miller & Costa, 1988; Schimmel & Budzig, 1977; Strniste & Smith, 1974; Wick & Matthews, 1991). However, the reagents designed for these studies have in almost all cases introduced the cross-links by formation of covalent carbon-carbon or carbon-heteroatom bonds. A number of disadvantages are inherent to these organic-based strategies. For example, the protocols typically require the introduction of highly reactive species, either by addition of a reactive chemical such as formaldehyde, (Renz, 1983; Renz & Kurz, 1984) or the in situ generation of a reactive intermediate through photochemical reactions (Meffert & Dose, 1988; Wick & Matthews, 1991, Frantz & O'Halloran, 1989; Peak et al., 1987). Because of the extreme reactivity of these species, the specificity of the reactions tends to be fairly limited. A second problem is that the chemical bonds mediating the cross-links are usually stronger than the linkages within the individual target biopolymers (i.e. peptide bonds or phophodiester linkages). Reversal of the cross-links is consequently difficult to achieve while maintaining the structural and functional integrity of the target molecules. In this latter regard, some improvement in reversibility has been accomplished by incorporation of a readily cleavable function such as a disulfide bond or vicinal diol into the cross-linker reagents (Kamp, 1988), but use of these reagents nevertheless results in the irreversible modification of the molecules of interest (Mauk & Mauk, 1989).
The manipulation of inorganic reactivity presents a solution to this problem. Many metal ions are capable of forming very strong coordinate bonds with the ligands present in proteins and nucleic acids, and metal ion-mediated cross-linking reactions have been reported in a number of instances (Miller & Costa, 1988; Mauk & Mauk, 1989; Ciccarelli et al, 1985; Kasselouri & Garoufis, 1990; Kosti'c, 1988; Miller & Costa, 1989). However, despite the very high formation constants which are frequently observed in certain coordination complexes of this sort, the coordinate bonds of a select group of metal complexes, particularly those of Hg(II), are nevertheless kinetically labile in some cases, and consequently are susceptible to competition by other ligands (Basolo & Pearson, 1960). Because they exhibit this combination of thermodynamic stability and kinetic lability, judicious selection of coordination complexes with the necessary thermodynamic and kinetic properties can provide the basis for crosslinking strategies which require the formation of very strong but reversible linkages.
The high affinity of complexes of the late transition metals for biological ligands such as the aromatic nitrogen atoms of the nucleobases and sulfur-containing sidechains in proteins has been exploited for the formation of strong covalent crosslinks in several in vivo studies. This application of transition metal chemistry has the major advantage that while some coordinate linkages are very stable, they can be nevertheless susceptible to exchange reactions with competing ligands, providing a means for reversal under mild conditions which do not cause the disruption of the biopolymer structures or leave residual covalent modifications.